Murine studies indicate that tumor associated myeloid cells are a legitimate therapeutic target in solid tumors. Studies in ovarian cancer have demonstrated an abundant population of tumor associated myeloid cells termed vascular leukocytes (VLCs). VLCs are highly immunosuppressive and proangiogenic, secreting numerous angiogenic factors to promote tumor growth. Therapies targeting VLCs potently restrict angiogenesis and inhibit ovarian cancer growth. Unfortunately the therapeutics used in animal models have limited use in humans due to significant side effects associated with the lack of specificity. We have therefore developed novel cytotoxic nanoparticles (G5-MTX-Nps) which are highly specific for VLCs. Because of their specificity, these Nps are expected to have limited side-effects. We therefore propose (1) to test G5-MTX- Nps as a novel anti-VLC, anti-angiogenic therapeutic. We will perform these studies using both murine ovarian tumor models, and human tumor models using freshly isolated human VLCs. Other anti-angiogenic therapies, such as anti-VEGF antibodies and VEGFR2 receptor tyrosine kinase inhibitors, have demonstrated clinical activity in ovarian cancer. Unfortunately patients on these therapies experience rapid relapses. Interestingly, relapses to anti-VEGF targeted therapies have been reported to be secondary to tumor recruitment of VLCs. We therefore hypothesize that elimination of VLCs from the ovarian cancer microenvironment can overcome resistance to anti-VEGF therapy. We therefore propose (2) to determine if cytotoxic G5-MTX-Nps targeting VLCs can overcome resistance to antiangiogenic therapy. We will perform these studies using three models of anti-VEGF resistance: ID8 murine ovarian tumors which progress through anti-VEGF therapy, ID8-VEGF tumors which express increased levels of VEGF, and ID8-VEGF-BDef tumors which recruit large numbers of tumor VLCs. In addition, we will use human tumor models using freshly isolated human tumor VLCs. Finally, VLCs are tightly associated with tumor vasculature. Interestingly, cancer stem cells (CSC) are also tightly associated with the tumor vascular, receiving essential survival cues from the tumor endothelial cells. In fact anti-angiogenic therapy can lead to a reduction in CSC via this loss of endothelial provided survival factors. We have preliminary evidence that VLCs also provide important growth signals for CSC. We therefore hypothesize that anti-VLC Np therapy will restrict CSC growth. Restriction of CSC growth may occur both via the loss of VLC produced growth factors and via the anti-angiogenic effects on tumor vasculature. We therefore propose to (3) to determine the impact of G5-MTX-Nps therapy on ovarian CSC survival and proliferation, both in vitro and in vivo. We will test these using in vitro sphere assays with primary human ovarian CSC as well as in mouse models of ovarian cancer stem cells, and human CSC xenografts.